Demonstration of cathepsin B in the rat kidney using fluorescence histochemistry

The procedure of mounting freeze-dried sections with celloidin was adapted for the fluorescent-histochemical demonstration of cathepsin B in the rat kidney. A good localization of reaction products was shown in freeze-dried, 5-micron sections which had been mounted free floating with 1.5% celloidin solution on albuminized slides. Using this procedure, the reaction products were localized in the lysosomes, particularly those of the convoluted proximal tubule.     

Sectioning Tough Stems Infiltrated with Polyethylene Glycol

Herbaceous stems eventually become too tough for paraffin sections. Alternative celloidin and plastic sections are laborious and expensive to make. Sticky or corrosive sap precludes fresh sections. These restrictions have been circumvented with a new method.     

Embedding with Low Viscosity Nitrocellulose

The gelation and cutting of embedding masses of low viscosity nitrocellulose (L. V. N.) and of celloidin were compared. L. V. N. forms a firmer mass which can be cut into thinner sections than celloidin. It tolerates considerable water (up to 6%) in a solvent system of alcohol-water-ether, thereby permitting the use of 95% alcohol instead of absolute for making up the solution. The fluidity of its solutions permits transfer of tissue directly from alcohol-ether to a 20% embedding solution. Faults of L. V. N. are: the nitrated cotton lint contains some grit (hence its solution should be allowed to settle), the sections cut from it are somewhat more easily torn than celloidin sections, and it is sufficiently soluble in absolute alcohol to preclude the use of this fluid in handling sections.     

Embedding with Low Viscosity Nitrocellulose

The gelation and cutting of embedding masses of low viscosity nitrocellulose (L. V. N.) and of celloidin were compared. L. V. N. forms a firmer mass which can be cut into thinner sections than celloidin. It tolerates considerable water (up to 6%) in a solvent system of alcohol-water-ether, thereby permitting the use of 95% alcohol instead of absolute for making up the solution. The fluidity of its solutions permits transfer of tissue directly from alcohol-ether to a 20% embedding solution. Faults of L. V. N. are: the nitrated cotton lint contains some grit (hence its solution should be allowed to settle), the sections cut from it are somewhat more easily torn than celloidin sections, and it is sufficiently soluble in absolute alcohol to preclude the use of this fluid in handling sections.     

Rapid Nissl Staining for Frozen Sections of Fresh Brain

Working with X-ray film autoradiography of soluble isotopes, we needed a staining technique for the localization of nuclei in frozen sections of fresh brain. We have found no Nissl staining method in the literature concerning autoradiography specially recommended for this purpose, nor have we found in handbooks on staining a Nissl method clearly recommended for unfixed, frozen sections of brain. The methods described are intended for paraffin or celloidin sections, and require fixation of brain before sectioning (which must be avoided when working with soluble isotopes). Because autoradiography is a time-consuming method, any technique which shortens time needed for the overall procedure is welcome. Most Nissl techniques described in the literature require long preprocessing of the tissue. We found two rapid methods, described by Humason (1967) and LaBossiere and Glickstein (1976), but their application to frozen sections did not give good results. After trials with several types of techniques, we succeeded in developing two Nissl modifications with slightly different qualities, one of 12 min and the other of 2-3 h. The longer method includes conventional steps in staining; the shorter method does not include fixation or lipid extraction. These methods were applied to 20-60 μm brain sections cut in the cryostat at -10 to -12 C and dried on gelatinized slides.     

The Rapid Preparation of Celloidin Serial Sections Following India Ink Injections

For the study of capillary penetration in the central nervous system of the chick embryo, following India ink injections, celloidin serial sections are superior to those prepared by the paraffin technic. The celloidin sections are arranged on a moist cigarette paper mat, which when filled is inverted and applied to a microscope slide so that the sections contact the glass surface. Subsequent to dehydration and clearing the sections are isolated on the slide by peeling off the cigarette paper. Forty-five minutes are required to prepare a slide of thirty sections from the time the block is trimmed until the cover slip is mounted with Clarite.     

Basic Dye Counterstaining of Sections Impregnated by the Golgi-Cox Method

Celloidin blocks of Golgi-Cox impregnated material are cut at 50 μ, the sections collected in 70% alcohol, transferred to a 3:1 mixture of absolute alcohol and chloroform for 2 min, and then stored in xylene or toluene for at least 3 min, or up to 2 wk until processed further. Mounting is done on glass slides which have been coated with fresh egg albumen diluted in 0.2% ammonia water (or a 0.5% solution of dry powdered egg albumen) and then dried at 60°C overnight. For attachment to these coated slides, sections are first soaked for 2-3 min in a freshly prepared mixture of methyl benzoate, 50 ml; benzyl alcohol, 200 ml; chloroform, 150 ml; and then transferred quickly to the slides by means of a brush. After 2-3 min the chloroform evaporates and the celloidin softens. The slides are then immersed in toluene which hardens the celloidin and anchors the sections to the slides. Alcohols of descending concentrations to 40% are followed by alkalinizations, first in: absolute alcohol, 40 ml; strong ammonia water 60 ml, for 2 min, then in: absolute alcohol, 70 ml; strong ammonia water, 30 ml, for 1 hr. Excess alkali is then removed by 70% and 40% alcohol, 2 min each, and a 10 min wash in running tap water. Bleaching in 1% Na₂S₂O₃, for 10 min and washing again in tap water for 10 min completes the process preliminary to staining. The preparations are then stained for 90 min in an aqueous solution of either 0.5% cresylecht violet, neutral red, or Darrow red, buffered at pH 3.6. Dehydration and differentiation in ascending grades of alcohol, clearing with toluene or xylene, and applying a cover glass with a mounting medium having a refractive index of about 1.61 completes the process.     

Notes on Technic: A Device for Centering Tissue Blocks on the Porter-Blum Microtome

A method of double embedding fixed tissues in 3% low viscosity nitrocellulose and paraffin is described. Five percent phenol in 80% alcohol during dehydration and 5% glycerin in the nitrocellulose solutions enhance cutting qualities. A modified Ruyter's solution is used to flatten sections. After a section is aflixed to a slide, it is passed through chloroform and acetone to remove the paraftin and celloidin. A 1% celloidin dip insures adherence of the seaion to the slide. Slides are stored in 70% alcohol until they are to be stained. Following staining and dehydration in graded alcohols, clearing should be done in a 1: 3 mixture of terpineol and toluene.     

Plastic Embedding of thick Celloidin Sections of nerve Tissue

A method is described for mounting Golgi-impregnated and Weigert-stained thick celloidin sections of brain and spinal cord in transparent plastic. Finished mounts have good optical properties and are suitable for macroscopic and microscopic observation. The durability of such preparations makes them superior to similar material prepared by the more conventional methods. Holes of suitable size were cut in matrices of 2.5 × 5 × 3/16 inches Plexiglas. Ward's Bio-plastic was used to form a base for the holes and also as the embedding medium for the sections. Plate glass formed a working substrate and gave a polished surface to the plastic base and later to the top of the preparation. For Golgi material (200μ) the celloidin was removed by dioxane. A dioxane-plastic bath preceded plastic embedding. For Weigert material (30-40μ) celloidin was not removed due to fragility of sections. Prior to plastic embedding, they were subjected first to benzol and then to a benzol-plastic bath.     

Double Embedding Again

A method of double embedding fixed tissues in 3% low viscosity nitrocellulose and paraffin is described. Five percent phenol in 80% alcohol during dehydration and 5% glycerin in the nitrocellulose solutions enhance cutting qualities. A modified Ruyter's solution is used to flatten sections. After a section is aflixed to a slide, it is passed through chloroform and acetone to remove the paraftin and celloidin. A 1% celloidin dip insures adherence of the seaion to the slide. Slides are stored in 70% alcohol until they are to be stained. Following staining and dehydration in graded alcohols, clearing should be done in a 1: 3 mixture of terpineol and toluene.     

A Teflon Disk for Processing Loose Serial Celloidin Sections

Celloidin sections are routinely used for Nissl, Golgi, or Golgi-Cox staining (e.g., Glaser and Van der Loos 1981) when sections thicker than 30 μm are required. In spite of the advantages of the celloidin method (see Voogd and Feirabend 1981, Buschke 1979), processing free-floating serial sections of celloidin embedded material, which may often be preferred, is not very convenient.     

Freeze substituted tissue in 5'-nucleotidase histochemistry. Comparative histochemical and biochemical investigations

The suitability of freeze-substitution in n-butanol and paraffin embedding of tissues for the histochemical demonstration of 5'-nucleotidase was investigated and compared with commonly used preparation techniques, such as fresh frozen sections and cryostate sections of cold formalin and glutaraldehyde-fixed rat liver. The influences of each step of the preparation techniques on the enzyme activity were controlled by a quantitative radiochemical assay. Freeze substitution was revealed to be superior to the commonly used preparation techniques with respect to: 1) high sensitivity and specificity of the histochemical 5'-nucleotidase reaction (this is based on the fact that incubation media with very low lead concentrations (0,6 mM/1) can be used); 2) excellent morphological appearance of the tissues showing cytological details of enzyme localization; 3) unlimited storage of the tissue materials and ease of sectioning.     

A new technique for staining sections and recovering celloidin film in high resolution autoradiography

Ultrathin sections are stained immediately after cutting by placing them in contact with staining solution and then placed on a slide covered by a celloidin film. This method largely avoids precipitates of heavy metals. The recovering of celloidin film is improved using a stainless steel basket. This technique is far more reliable than that involving use of a filter paper.     

Mounting Frozen Sections with Gelatin

Frozen sections, 15-50 µ thick, are soaked for 5 minutes or longer in a mixture of equal parts of 1.5% aqueous gelatin and 80% alcohol, and teased onto a slide. After allowing excess fluid to evaporate, sections will be moist and can be blotted with filter paper that may require dampening with 95% alcohol. Immersed in 95% alcohol, the remaining gelatin will congeal, anchoring the section to the slide. If necessary, the sections can subsequently be coated with celloidin.     

A Simplified and Convenient Method for the Double-Embedding of Tissues

A method for embedding tissues with a celloidin-paraffin combination is presented. The essential features of the process depend upon (1) a thorough infiltration of the specimen with celloidin of low concentration, and (2) the subsequent impregnation of both the specimen and the celloidin with paraffin.

The methods for sectioning, and the removal of the embedding agent are given.

The chief advantages of this method are: the preservation of all of the advantages of celloidin embedding but with a great saving of time, and greater convenience of storage; the cutting of thin sections (2μ for many types of tissues); it is useful for embedding specimens for which neither pure paraffin nor pure celloidin are entirely satisfactory, i.e. those containing tissues differing in density.     

A Protargol Method for Staining Nerve Fibers in Frozen or Celloidin Sections

Extensive experimentation with protargol staining of neurons in celloidin and frozen sections of organs has resulted in the following technic: Fix tissue in 10% aqueous formalin. Cut celloidin sections IS to 25 μ, frozen sections 25 to 40 μ. Place sections for 24 hours in 50% alcohol to which 1% by volume of NH₄OH has been added. Transfer the sections directly into a 1% aqueous solution of protargol, containing 0.2 to 0.3 g. of electrolytic copper foil which has been coated with a 0.5% solution of celloidin, and allow to stand for 6 to 8 hours at 37° C. Caution: In this and the succeeding step the sections must not be allowed to come in contact with the copper. From aqueous protargol, place the sections for 24 to 48 hours at 37° C. directly into a pyridinated solution of alcoholic protargol (1.0% aqueous solution protargol, 50 ml.; 95% alcohol, 50 ml.; pyridine, 0.5 to 2.0 ml.), containing 0.2 to 0.3 g. of coated copper. Rinse briefly in 50% alcohol and reduce 10 min. in an alkaline hydroquinone reducer (H₃BO₃, 1.4 g.; Na₂SO₃, anhydrous, 2.0 g.; hydroquinone, 0.3 g.; distilled water, 85 cc; acetone, 15 ml.). Wash thoroly in water and tone for 10 min. in 0.2% aqueous gold chloride, acidified with acetic acid. Wash in distilled water and reduce for 1 to 3 min. in 2% aqueous oxalic acid. Quickly rinse in distilled water and treat the sections 3 to 5 min. with 5% aqueous Na₂S₂O₃+5H₂O. Wash in water and stain overnight in Einarson's gallocyanin. Wash thoroly in water and place in 5% aqueous phosphotungstic acid for 30 min. From phosphotungstic acid transfer directly to a dilution (stock solution, 20 ml.; distilled water, 30 ml.) of the following stock staining solution: anilin blue, 0.01 g.; fast green FCF, 0.5 g.; orange G, 2.0 g.; distilled water, 92.0 ml.; glacial acetic acid, 8 ml.) and stain for 1 hour. Differentiate with 70% and 95% alcohol; pass the sections thru butyl alcohol and cedar oil; mount.     

Rapid method of silver nitrate impregnation of elements of the peripheral nervous system suitable for celloidin and paraffin sections

According to the method of neural elements impregnation in the authors' modification, the object is fixed for 6-12 h in Lillie fluid cooled to 4 degrees C. Then the object is kept under tap water for 2-6 h. Frozen sections are prepared and kept in pure pyridine for 1-6 h. When the sections are embedded into paraffin or celloidin, they are put into alcohol solutions gradually decreasing their concentration until water is reached, then put into pyridine. In order to remove cellulose, the celloidin sections are treated in 3 portions of pyridine (in the 1st and 2nd-for 10 min, and in the 3d-for 6 h). Then they are washed under tap water for 2-4 h and in distilled water for 30-40 min. Further treatment is performed according to the methods by Bielschowsky - Gros, Kampos or Rasskazova. Excess silver is removed by treating the sections in 2% ammonium persulfate under the microscope control (the process is stopped by putting the sections into 7% sodium hyposulfate for 10 min). Then the sections are treated in 0.1% aurum chloride, in 5% hyposulfite to reveale the tissue background [corrected] and by means of routine histological techniques either after Brashet, Hale, PAS-positive reaction or other methods applied after fixation in Lillie fluid.     

The Preparation of Sections of Mineralized Tissue Suitable for the Demonstration of Alkaline Phosphatase

Blocks of molar teeth and bisected knee joints from rats of 7-21 days were embedded, without previous decalcification, in tropical grade ester wax. Serial sections were cut at settings of 3-25μ on a base sledge microtome equipped with a Jung extra hard steel knife with a tool-edged profile. The sections were supported with Sellotape during actual cutting and were then coated with a 2% celloidin solution. Chloroform was used to free the sections from the Sellotape. The distribution of alkaline phosphatase activity in the knee joints and teeth was demonstrated in these sections with the coupled-azo dye technique of Gomori, using Brentamine fast red T.R. salt.     

Factors which Influence the Latent Image in Autoradiography

Sections treated with N-ethyl-maleim'ide, a sulfhydryl blocking reagent, exhibit similar grain counts over nonradioactive and S-labeled tissues. This reveals that no chemo-graphic effects were produced by sulfhydryl groups in tissues fixed and prepared in the manner described. It was noted, however, that background count over tissue sections is consistently tower than over adjacent him, indicating perhaps the existence of substances in tissues which interfere with the production of the latent image. These effects have been eliminated by coating the tissues with celloidin. Celloidin coating can be used for most emitters including S but not for tritium. A special method for tritium, not involving celloidin coating is given.     

Factors which Influence the Latent Image in Autoradiography

Sections treated with N-ethyl-maleim'ide, a sulfhydryl blocking reagent, exhibit similar grain counts over nonradioactive and S-labeled tissues. This reveals that no chemo-graphic effects were produced by sulfhydryl groups in tissues fixed and prepared in the manner described. It was noted, however, that background count over tissue sections is consistently tower than over adjacent him, indicating perhaps the existence of substances in tissues which interfere with the production of the latent image. These effects have been eliminated by coating the tissues with celloidin. Celloidin coating can be used for most emitters including S but not for tritium. A special method for tritium, not involving celloidin coating is given.